The Involvement Of SPL In The Effect Of Terminal Shoot Maturity On Litchi(Litchi Chinensis Sonn.) Flowering

Litchi flowers in the terminal shoots after exposure to sufficient cold temperature,but the immature shoots are not competent to flower even in the presence of low temperature induction.SPL(SQUAMOSA promoter-binding protein like)plays an important role in age-dependent flowering pathway,but the involvement of SPL in shoot maturity-mediated litchi flowering in adult litchi plants is unclear.In the present study,litchi plants with the terminal shoot at mature,turning and extending stage were treated with 60-days of cold,and then the flowering ability was evaluated.We analyzed the expression pattern of SPL genes in litchi leaves at different stages,identified the target genes of SPL by transient expression system and Yeast-one-hybrid(Y1H),and illustrated the SPL-dependent regulatory pathway in litchi flowering.We also sampled shoot apical meristem(SAM)tissues to compare the varied role of SPL in leaves and SAM.The main results are mentioned as follow:1.To study the role of Lc SPLs in SAMs in litchi flowering,we sampled the SAMs from litchip plants in orchard in two successive years.Both plants with mature and extending terminal shoots could form “whitish millets”,but the ratio of flowering shoots highly varied(5.18% and 37.43%,respectively).Lc FT1,Lc FT2,Lc SPL1,Lc SPL3 and Lc SPL4 were highly expressed in mature leaves,but Lc SPL6 had the highest expression level in SAMs and declined expression during the cold induction.Lc TFL1,a putative flowering repressor,showed similar expression pattern with Lc SPL6.Furtherly,Lc SPL6 were found to bind to the promoter of Lc TFL1 gene to activate its expression.According to Y2 H analysis,both Lc FT1 and Lc TFL1 can interact with Lc FD protein.2.In the green house,potted litchi plants with the terminal shoot at mature,turning and extending stage were treated with 60-days of cold(15/10?),and then were transferred into room temperature(25?).During the cold treatment,the growth of plants slowed down,and there were no significant changes in length,width and SPAD values of leaves in terminal shoots.After the cold induction,the ratio of flowering shoots in mature,turning and extending plants are 84.62%,16.11% and 14.51%,respectively.However,the ratio of flowering shoots in the group of turning plants increased to 66.95% after spray of sucrose(15 g/L).These results indicated that mature terminal shoots are crucial for litchi flowering,and sucrose exhibited important role in shoot maturity-dependent litchi flowering.3.During the cold induction,Lc SPL1,Lc SPL3,Lc SPL4,Lc SPL10 were highly expressed in leaves from terminal shoots of mature plants in comparison to the turning and extending plants.The expressions of these Lc SPLs were induced by cold temperature and acchived the peak level at “whitish millets” stage(floral initiation).However,Lc SPL6,Lc SPL13,Lc SPL14,Lc SPL17 exhibited the oppsite expression pattern.During the cold induction,the expression of Lc FT1 and Lc FT2 were significantly increased in mature plants,but these increases were not observed in extending plants.According to the analysis of Y1 H and Dual Luciferase Reporter System,Lc SPL3 and Lc SPL10 were found to bind to the promoter of Lc FT1 to activate its expression.However,only overexpression of Lc SPL10 genes in Arabidopsis could accelerate flowering.Taken together,Lc SPL1,Lc SPL3,Lc SPL4 and Lc SPL10 might be involved in shoot maturity-dependent litchi flowering,and Lc SPL3 and Lc SPL10 could regulate litchi flowering by direct targeting Lc FT1.Lc SPL6 and Lc TFL1 were suggested to be the flowering repressors in litchi by that Lc SPL6 mainly expressed in SAMs to target Lc TFL1.During litchi floral induction,the expression of Lc SPL6 and Lc TFL1 decreased to reduce the competition of binding of Lc FT1 and Lc FD,eventually accelerated flowering.